Preparation of test sample for immunological assay of pregnancy of mares

ABSTRACT

A process for the preparation of a sample to be tested by immunological assay for detecting serum gonadotrophin in blood of mares, for the purpose of diagnosing the mares pregnancy, which comprises adding an acid selected from the group consisting of sulfosalicyclic, perchloric, and metaphosphoric acids, to the blood, plasma, or serum of the mare to be diagnosed, at a concentration of at least 0.15 m-mol based on 1 ml of serum but below the saturation concentration, separating the resultant precipitate and recovering the supernatnat liquid, and a method diagnosis of pregnancy of a mare using such test sample.

United States Patent Matsushima Sanae et al.

[1 1 3,689,633 [451 Sept. 5, 1972 [54] PREPARATION OF TEST SAMPLE FOR IMMUNOLOGICAL ASSAY OF PREGNANCY OF MARES [72] Inventors: Matsushima Sanae, Tokyo; Tadashi Tezuka; Karneo Nakanowatari, both of Kawasaki-shi, all of Japan [73] Assignee: Teikoku Hormone Mfg. Co., Ltd-.,

Tokyo, Japan 22 Filed: Jan. 13,1969

211 Appl.No.: 790,868

[52] U.S. Cl. ..424/12, 260/112 R, 424/78, 424/ 100, 424/101 [51] Int. Cl. ..A23j 1/06, C07g 7/00, G01n 33/16 [58] Field of Search.....424/l2, 100, 10l;260/112 R, 260/112 B [56] References Cited OTHER PUBLICATIONS Leclercq(l), Vet. Bull. Vol. 34, (1964) p 622 No. 3916 Wide, Nature, Vol. 198, June 8, 1963 pp 1017- 8 Richards, Nature, Vol. 215, Sept. 16, 1967 pp 1280- 1 Leclercq (2) Acd. Vet. de France Bull. Vol. 37, 1964 pp 47 53 Bourrillon (1), Bull. Soc. Chim Biol Vol. 39, 1957 pp 1119- 1128 Bourrillon (2) Bull. Soc Chirn Biol Vol. 40, 1958 pp 87- 97 Chem Abs. Vol. 48, 1954 p 13747 vol. 51, 1957 p 8863 Rimington, Biochem J. Vol. 38, 1944 pp 54- 60 Primary Examiner-Albert T. Meyers Assistant ExaminerA. P. Fagelson Attorney-Shennan & Shalloway ABSTRACT A process for the preparation of a sample to be tested 1 Claim, No Drawings PREPARATION OF TEST SAMPLE FOR IMMUNOLOGICAL ASSAY OF PREGNANCY OF MARES.

This invention relates to an improved process for making the sample to be tested (hereinafter called test sample) for diagnosing pregnancy of mares by immunological assaying of gonadotrophin contained in the blood, plasma or serum of pregnant mares. The invention furthermore relates to an improved method for diagnosing pregnancy of mares, and also to the set of chemicals for diagnosing pregnancy of mares.

A pregnant mare, secretes pregnant mare serum gonodotrophin (hereinafter to be abbreviated as PMS), which is one of the glycoproteins, from placenta. This hormone becomes observable in the mares blood approximately thirty days after fecundation, and its concentration in the blood reaches the maximum during the period of approximately 40 90 days after fecundation. Therefore an assay of PMS in mares blood enables early diagnosis of pregnancy. This method has been considered to be the most reliable diagnostic method for that purpose, but is subject to such a drawback that it is too complex for clinical diagnosis, because the necessity for utilizing biological activity of PMS requires living laboratory animals.

Recently, however, a method for assaying PMS by an immunological means was discovered, and thereby an easy assaying in short period became possible. However, by that method a correct diagnosis is difficult if the blood, plasma, or serum of a mare is used as the teat sample as it is, because the same contain blood components which interfere with the assaying reaction (presumably the proteinous components other than PMS) at high concentrations and consequently the accuracy of the reaction is impaired. In order to avoid such inaccuracy, it was proposed to isolate serum from the blood to be assayed, and to use the serum, which is subsequently diluted to the critical concentration to eliminate the action of objectionable components, for example, proteins, as the test sample. Also, as an alternative, it is known to add an organic solvent such as alcohol or acetone to the serum to precipitate PMS, and to use as the test sample the extract obtained by extracting the precipitate with isotonic sodium chloride solution. However, in the former method the correctly positive reaction result may not be obtained due to low PMS content in the test sample, since the PMS concentration in the sample also is reduced by the dilution. Whereas, the latter method is unsuitable for clinical use since it involves complex procedures.

It has now been discovered that, among the various protein precipitation or deproteinizing agents, particularly sulfosalicylic acid, perchloric acid, and metaphosphoric acid give favorable results. That is, when such an acid is added to blood, plasma or serum of a pregnant mare (hereinafter the substances will be collectively referred to as the serum) at a specific concentration, the blood components interfering with the haemagglutination inhibition reaction of gonadotrophin in the serum, such as proteins, can be selectively precipitated, without affecting the gonadotrophin. It has furthermore been discovered that such a test sample for diagnosis of pregnant mare utilizing the haemagglutination inhibition reaction of PMS can be prepared with simple operation, without diluting the serum.

2 Accordingly, therefore, an object of the invention is to provide a process for the preparation of test sample for diagnosis of pregnant mare by means of haemagglutination inhibition reaction of PMS, which can be easily practiced by a veterinary surgeon on the spot.

Another object of the invention is to provide a process for the preparation of test sample which is valuable for diagnosis of pregnant mare by means of haemagglutination inhibition reaction of PMS, which enables determination of mares pregnancy with very high reliability.

Still another object of the invention is to provide an improved method of diagnosis of pregnant mare by means of haemagglutination inhibition reaction of PMS, which enables veterinary surgeons to determine if the mare is pregnant with ease and high reliability at the spot of diagnosis, and also to provide as set of diagnostic chemicals to be used in that method of diagnosis.

The foregoing objects of the invention are accomplished by the process for the preparation of sample to be tested by immunological assay for detecting serum gonadotrophin in blood of mares, for the purpose of diagnosing the mares pregnancy, which comprises adding an acid selected from the group consisting of sulfosalicylic, perchloric, and metaphosphoric acids, to the blood, plasma or serum of the mare to be diagnosed, at a concentration of at least 0.15 m-mol based on 1 ml of the serum but below the saturation concentration, separating the resultant precipitate and recovering the supernatant liquid.

In accordance with the invention, the blood, plasma or serum of the mare to be diagnosed can be used. For taking blood from the mare, it is important to select the period during which the PMS concentration in the mare s blood reaches its maximum, in order to improve the accuracy of diagnosis. As aforesaid, the highest concentration is normally obtained during approximately 40 to 90 days after fecundation. Accordingly, in accordance with the invention it is preferred to use blood, plasma or serum of a mare, taken on the 40th to th day from the last mating. It is of course possible to use blood, plasma or serum of a mare taken on the 30th to 40th day from mating, if an early diagnosis is aimed at. However, since the reliability of the diagnosis in that case is somewhat impaired, a re-diagnosis at a later date may be required. The amount of blood required is preferably 2 10 ml per one diagnosis, for easy operation.

According to the invention, an acid selected from the group consisting of sulfosalicylic, perchloric and metaphosphoric acids is added to the mares blood, plasma or serum, at a concentration of at least 0.15 mmol based on 1 ml of serum, but below saturation point. With the addition of such a specific acid in the specific amount, it is possible to selectively precipitate the blood components interfering with the haemagglutination inhibition reaction of gonadotrophin, such as proteins other than PMS, without precipitating the PMS contained in the serum.

It has been known that addition of an organic acid such as trichloroacetic, sulfosalicylic, picric, or tannic acid; an inorganic acid such as phosphotungstic, phosphomolybdic, tungstic, and metaphosphoric acid; or a heavy metal salt such as zinc chloride, copper sulfate, lead acetate, Reineckes salt, etc. to an aqueous solution of proteinous substance is useful for precipitation of the proteinous substance. However, it has been discovered that, among the above many deproteinizing agents, particularly sulfosalicylic, perchloric, and metaphosphoric acids only can selective precipitate the objectionable components (i.e. proteins other than PMS), without affecting PMS, at relatively wide concentration ranges. In fact, deproteinizing agents outside the scope of this invention are unsatisfactory in that either they precipitate PMS and the objectionable components simultaneously, or cannot efiectively precipitate the objectionable components. Furthermore, for some deproteinizing agents which exhibit neither of the above objectionable activities, the effective concentration range is extremely narrow and clinically impracticable.

The above statement is substantiated by the data given in Table 1 below, which are obtained by subjecting test bodies, prepared by adding various deproteinizing agents to a pregnant mares serum at various concentrations, to a haemagglutination inhibition reaction test.

METHOD OF TESTING Each 2 ml of a non-pregnant mares serum, and of the same serum in which 12 international unitsof PMS per 1 ml of the serum had been dissolved, were taken was taken with a pipette, and neutralized to pH 5.0 8.5 with N.NaOl-l. The prepared test samples were subjected to the haemagglutination inhibition reaction test in the following manner.

Into a small test tube of approximately 1 cm in diameter, 0.3 ml of a diluted solution of an antiserum which will be fully explained later was taken, and to which 0.1 ml of the test sample and adrop (approximately 0.07 ml) of PMS-sensitized blood cell suspension (which will be specified later) were added, well shaken and allowed to stand. After allowing to stand for approximately 2 hours, the bottom of the test tube was observed. When PMS was present in the test sample as dissolved in the serum, the PMS and the antibody were bound, but bonding of PMS-sensitized blood cells with the antiserum, i.e., agglutination of blood cells was inhibited, and consequently the blood cells were precipitated, forming a ring. in contrast thereto, when PMS was absent in the test sample, the agglutination of perirnents, in which the evaluation of is given to PMS-sensitized blood cells was not inhibited, and consequently a uniform mat covering the entire bottom of the test tube was formed.

Table 1 given below shows the results of above exclearly ring-formed precipitate, 1*: is given to ambiguously ring-formed precipitate, and to the matlike precipitate. Also in the column on properties of su matant liquid, the protein concentration was deterinto small test tubes, and to each Of the tubes 2 ml Of mined Lawry's reaction (Lowry OH,

the deproteinizing reagent solution of the concentration given in the table were added, followed by thorough agitation. Each sample was then centrifuged for 30 minutes at a rate of approximately 3,000 rpm.

Rasebrough, A.L. Farrand & R.I. Randall; J. Biol. Chem. 193. 265. i951), and expressed by absorbance at a wavelength of 750 mu. The concentrations marked with sign were those in the supernatant liquids of The coagulated protein was precipitated and removed. which pH was controlled with N. HCl after addition of The transparent or slightly turbid supernatant liquid the reagent solution.

TABLE 1 Reaction Deproteinizing reagent Properties of supernatant liquid Serum con- Concen- Protein con- Sermn containlng no Type tratlon Color pH eentration taining PMS PMS Sulfosalieyliohcld 1. 2M Slightly turbid o. 3 1,390 0.8 0. 5 1,325 A 1.1 855 0. 2 Red, slightly turbi 1.4 940 0. 1 Red, turbi 2. 3 800 :1: 0. 05 Red, turbid s. a (x10 1, 710

Metaphosphorie acid 3. 2 M Slightly turbid 0. 9 (X10) 1, 125 =1:

Perchlorie acid 1, 005

- 600 550 OA 3 850 0.2 Slightly turbid 0. 97 (X10) 2, 850

0. 1 Yellow, slightly turbid 2. 57 (X100) 740 Triehloroacetic acid 290 292 403 g 1,110

0. 1 Yellow, slightly turbid 3. 57 (X100) 1, 011 0.05 -....do 4.67 (X100) 1,055

Phosphotungstic aeid l Phosphomolybdic acid .Q

Tannin acid 1% 5. 0 100 "1% Brown, turbid- 3. 1 (X100) 1, 840

Zinc chloride TABLE 1 Deproteinizing reagent Concen- Type tration Color Picrlc acid Properties of supernatant liquid Reaction Serum containing no PMS Serum containing PMS Protein concontratlon Measurement impossible weewweoaw SSEEZEE (X10) 1.340 (x10) 1,620 (x10 2,050 (x10) r300 Q... i" 9. 99. 5 eiee pppppp Bluish green Copper sulfate eeeeeewwo Measurement impossible M Yellow, slightly turbid.

Lead acetate Reineckes salt From the results of above Table l, the following is' evident. That is, (i) that sulfosalicylic acid, perchloric acid and metaphosphoric acid employed in the invention can eliminate the objectionable components without precipitating PMS, over a wide concentration range from 0.15 m-mol per 1 ml of serum to the saturation concentration, and thereby enable easy determination of mares pregnancy by haemagglutination inhibition reaction; (ii) that. an organic acid such as tannic acid precipitates PMS together with the objectionable components, and hence cannot be used in the preparation of test sample for haemagglutination inhibition reaction test; (iii) that heavy metal salts such as zinc chloride, copper sulfate, lead acetate and Reineckes salt cannot remove objectionable proteins other than PMS even when used at high concentrations, and therefore the resultant supernatant liquids cannot remove objectionable proteins other than PMS even when used at high concentrations, and therefore the resultant supernatant liquids cannot be putto the haemagglutination inhibition reaction test; and (iv) that other acids such as phosphotungstic, trichloroacetic, phosphomolybdic acids, etc., are unsuitable for clinical application, because the concentration range at which they exhibit the selective precipitating activity for objectionable components other than PMS is extremely narrow.

Furthermore, in the definition of deproteinizing agent concentration given in this specification and claims, per 1 ml of serum means that when blood or plasma'is used, 1 ml of serum contained in the blood or plasma is used as the base. Normally blood contains approximately half the amount thereof of serum. Therefore 1 ml of serum roughly corresponds to 2 ml of blood. A plasma can be regarded as composed substantially of serum alone, without any practical inconvenience.

The preferred amount of deproteinizing agent to the serum in the elimination of objectionable varies depending on the type of deproteinizing agent employed, but is normally within the following ranges:

llll|+ lllllll lllllH- lllllll Amount 0.2 l.2 m-mol/l ml serum 0.2 3.2 m-mol/l ml serum 0.4 2.4 m-mol/l ml serum -Deproteinizing agent Sulfosalicylic acid Perchloric acid Metaphosphoric acid While blood, plasma or serum of marecan be used in the. present invention, serum is the most preferred, because of simplicity in handling and high reliability of diagnosis. Whenblood or plasma is used, use of a relatively larger amount of deproteinizing agent is recommended for improving the reliability.

As the deproteinizing'agent, sulfosalicylic acid and perchloric acidareparticularly valuable, because their aqueous solutions are stable and very convenient for clinical application. Whereas, aqueous solutions of metaphosphon'e acid are very unstable and change to aqueous phosphoric acid solutions having no deproteinizing action during a few days standing. Therefore, when metaphosphoric acid is used as the deproteinizing agent, the aqueous solution thereof must be prepared on the spot of diagnosis.

The deproteinizing agent employed in this invention an 50 is preferably formed into an aqueous solution in advance of actual usage.

Generally it is preferred for convenient use to prepare an aqueous solution of sulfosalicylic or perchloric acid having a concentration ranging from 0.15 mol'to the saturated concentration in advance, and to addto the serum 0.5 2 volume times, particularly equal volume, of the aqueous solution. Addition of a greater amount of the aqueous solution will unduly lower the PMS concentration in the test sample, and that of a less amount of the aqueous solution will render the elimination of precipitate difficult.

Separation of the precipitate can be effected by the means known per se, for example, centrifugation, filtration, or decantation after allowing to stand, centrifuging being preferred for quick operation.

Thus, in accordance with the invention, the test sample composed of supernatant liquid which contains PMS at a high concentration but substantially no com ponents which interfere with the haemagglutination inhibition-reaction of PMS can be easily obtained. In fact, in accordance with the invention, the necessary operation of a veterinary surgeon for the preparation of test sample is no more than the addition of an equal amount of deproteinizing agent solution to the mares serum and removal of the resultant precipitate from the system.

The obtained supernatant liquid is acidic due to the deproteinizing agent employed, which can be optionally neutralized with a caustic alkali solution. In that case, normally IN caustic soda aqueous solution is conveniently used. In the neutralizing procedure, a pH indicator may be added previously into the reagent for precise indication of neutralization point, to facilitate an easy and accurate. pH control of the supematant liquid, for example, pH to 8.5. As such an indicator, p-nitrophenol is preferably recommended.

The supernatant liquid as the test sample is then sub jected to the known haemagglutination inhibition reaction test. The amount of the test sample required for this test is in the order of 0.1 ml.

Furthermore, the invention provides a method of diagnosis of pregnancy of mares comprising mixing a test sample containing PMS but containing substantially no proteinous impurities, with antiserum-against PMS, mixing the resultant solution with PMS-sensitized blood cells, and allowing the solution to stand to perform the haemagglutation inhibition reaction test, which is characterized in that the test sample is obtained by the steps of adding an acid selected from the group consisting of sulfosalicylic, perchloric and metaphosphoric acids to blood, plasma or serum of the mare to be diagnosed, at a concentration ranging from 0.15 m-mol based on 1 ml of serum to the saturation concentration, separating the precipitate formed and recovering the supernatant liquid, and neutralizing the same with an alkali to a pH ranging from 3 9.5, particularly 5 8.5

The PMS-sensitized blood cells to be used in the haemagglutination inhibition reaction test is a substance itself known. It can be prepared, for example, by the method disclosed in Nature, Vol. 198. p. 1017 l 963 In one embodiment, a fixing agent such as formaldehyde, pyruvic aldehyde, glutaraldehyde, or hydrogen peroxide, is added to a suspension of sheeps erythrocytes, followed by incubation to fix the erythrocytes. The fixed erythrocytes are then treated with tannic acid or bis-diazotized benzidine, and to which a PMS solution is added to sensitize the erythrocytes with PMS.

- A specific example of preparing PMS-sensitized blood cells will be given hereinbelow.

EXAMPLE A Erythrocytes of a sheep are thoroughly washed with isotonic sodium chloride solution, and thereafter formed into a 8 percent erythrocyte suspension, To the suspension an equal amount thereto of 3 percent formalin is added, followed by heating to 37C. for approximately 24 hours to fix the erythrocytes. The fixed erythrocytes are washed and suspended in phosphoric acid buffersolution at a concentration of 8 percent. An equal amount of 1/20,000 tannic acid solution is added to the suspension, and the system is heated to 57 C. for 30 minutes, followed by washing with phosphoric acid buffer solution. The system is then suspended in the same buffer solution at a concentration of 8 percent.

To the suspension, then IU/ml of PMS solution is added, followed by 2 hours heating at 56 C. to complete PMS-sensitization. The system is finally washed with phosphoric acid buffer solution and is made into 2.5 percent PMS-sensitized erythrocyte suspension.

The obtained PMS-sensitized blood cell suspension can be stored in a cold place as it is, and optionally taken out for actual use. However, it is preferred for clinical use to keep it in the form of frozen and dried, powder product.

As already mentioned, only a drop (approximately 0.07 ml) of this PMS-sensitized blood cell suspension is required in one test. The suspension of the amount necessary for one to several tests can be enclosed in an ampule for convenient use by veterinary surgeons, together with an inert solid carrier, for example, a saccharide such as lactose, sucrose, sorbitol, etc. and frozen and dried.

The antiserum against PMS, which is employed in the invention, is again a know substance itself, and can be prepared by the means known per se. The prepareation method again is described in the aforesaid Nature, Vol. 198,p. 1017 (1963).

EXAMPLE B For example, 2,000 5,000 IU/ml of PMS solutionis mixed-with an equal amount of an adjuvant, such as Romons or Freunds adjuvant. Then a mature rabbit is immunized by hypodermic injection of the mixture ob tained in the above, three to four times every other week. The rabbit blood. is extracted and the serum therefrom, is separated, and the antiserum against PMS can be obtained. I

It is preferred to use the antiserum obtained as in the above in an amount as much as 1.5 4 times the corresponding amount to PMS-sensitized blood cells. For this purpose, the rabbits antiserum against PMS is multiply diluted with phosphoric acid buffer solution to form various multiply diluted solutions, and each 0.3 ml of the dilute solutions is taken into a small test tube. Into each test tube then a drop (approximately 0.07 ml) of the PMS-sensitized blood cell suspension 1 is added, and the system is well shaken and left to stand for approximately 2 hours. Then the state of the aggregate formed at the bottom of test tube is observed. if evenly spread precipitates covering the entire bottom of the tube are observed, the state is judged to be agglutination, and when red, ring-formed precipitates are observed, the judgment of non-agglutination is given. Thus the maximum value of diluting multiple samples of the antiserum showing the perfect agglutination is determined, and 1/ 1,5 l/4 of the maximum value of diluting multiple samples is used as the antiserum concentration corresponding to the PMS-sensitized blood cell suspension employed in the experiment. Then the dilute solution having the so determined concentration is referred to as dilute solution of the antiserum.

This dilute solution of the antiserum can be stored in a cold place as it is, and optionally taken out at the required occasion. However, for convenient clinical application, it is preferred to insert the necessary amount of the dilute solution of the antiserum for single time usage, i.e., 0.3 ml, into an ampule together with an inert solid carrier, for example, saccharides such as lactose, sorbitol, etc., or an amino acid such as glycine,

sodium glutamate, etc., followed by freezing and drymg.

The diagnosis on pregnancy of mare utilizing the haemagglutination inhibition reaction of PMS is performed as follows 0.3 ml of the dilute solution of the antiserum is placed in a small test tube, 0.1 ml of the test sample and a drop of the PMS-sensitized blood cell suspension are added to the test tube, the mixture is shaken well, and is allowed to stand for approximately 2 hours. Then the inside bottom of the test tube is observed. When the test sample contains PMS dissolved therein, the PMS and antibody are bound and thereby the bonding of PMS-sensitized blood cells with the antiserum, i.e. agglutination of blood cells is prohibited. Accordingly the blood cells are precipitated in the form of a ring. In contrast, if the test sample no contains PMS, the agglutination of PMS-sensitized blood cells is not inhibited, and consequently an evenly precipitated blood cell aggregation is observed at the bottom of the test tube. PMS can be thus detected by the foregoing method. Also when determination of PMS is necessary, the test sample is multiply diluted, and the quantity of PMS is calculated from the maximum dilution ratio.

When the frozen and dried products of antiserum and PMS-sensitized blood cells are used, the following procedures are followed 0.1 ml of the test sample is added to the antiserum powder in the ampule, to dissolve the latter. Separately, 1 ml of a buffer solution of pH 5 9, inter alia, 6 8, is added to the PMS-sensitized blood cell powder in the ampule, to form the 3 suspension of the PMS-sensitized blood cells. Then 0.2 0.4 ml of this suspension is added to the test sample containing the antiserum dissolved therein.

In assaying PMS by means of the haemagglutination inhibition reaction as mentioned above, if the blood or the serum as extracted from a mare is used as the test sample, certain components in the blood inhibit the agglutination of PMS-sensitized blood cells. Consequently the blood cells are precipitated in a ring form even when the test sample is taken froma non-pregnant mare and no contains PMS, showing a positive reaction result. This inevitably invites erroneous diagnosis. However, in accordance with the present invention, the components causing such inconveniences are sufficiently removed from the test sample by the deproteinizing reagent specified in the foregoing. Accordingly, very accurate diagnosis on pregnancy of mare can be given, suing the supernatant liquid, which is separated from the objectionable components, as the test sample.

The reagents to be employed in the subject method of diagnosis are preferably prepared in the form of a set, for convenient use by clinical veterinary surgeons.

This invention also provides a set of diagnostic chemicals for pregnancy diagnosis of a mare of haemagglutination inhibition reaction of gonadotrophin in blood, plasma or serum of a pregnant mare,

which comprises an antiserum against PMS, and PMS- sensitized blood cells which are prepared by the steps of fixing blood cells with a fixing agent selected from the group consisting of formaldehyde, pyruvic aldehyde, glutaraldehyde and hydrogen peroxide, treating the fixed blood cells with an agent selected from the group consisting of tannic acid and bis-diazotized benzidine, and sensitizing the same with PMS, the set characteristically comprising a deproteinizing agent consisting of an aqueous solution of an acid selected from the group consisting of sulfosalicylic acid and perchloric acid, of a concentration ranging from 0.15 mol to saturated concentration.

The above-specified set can optionally include an aqueous solution of a caustic alkali such as caustic soda or caustic potash, at a concentration of 0.5 5N, as a neutralizing agent of the acid. Also when frozen-dried powder of PMS-sensitized blood cells is used, a buffer solution of pH 5 9, 2, preferably 6 8, can be included in the set for dissolving the powder.

EXAMPLE 1 A set of the following diagnostic chemicals was prepared A tube Frozen and dried product, 0.3 ml of the antiserum dilute solution prepared in accordance with Example B, put in an ampule together with 3 mg of lactose;

B tube Frozen and dried product, 0.35 ml of PMS-sensitized blood cell suspension obtained in Example A, placed in an ampule together with 20 mg of sucrose;

C tube 1.0 ml of a buffer solution, such as those described below, inserted in an ampule;

Types of buffer solutions 1. Phosphoric acid buffer solution Per ml of the solution Sodium hydrogenphosphate (Na i-IP0 Potassium dihydrogenphosphate (Kl-MPO,)

Sodium chloride (NaCl) 2. Boric acid buffer solution Per. ml of the solution Boric acid (H 4.4 mg Sodium borate (Na B O l 0H O) Sodium chloride (NaCl) 7.6 mg pH 8.2 8.3

3. Citric acid buffer solution Per ml of the solution Citric acid (C H.,(OH) (COOH) l 1.13 mg Potassium hydroxide (KOH) 8.53 mg D tube 2 ml of 0.3 mol concentration solution of sulfosalicylic acid, inserted in an ampule;

E tube 0.5 ml of lN-sodium hydroxide solution inserted in an ampule.

Usage of the set l 4 ml of blood, or 2.0 ml of plasma or serum was extractedfrom a 6 yrs. old percheron mare 49 days after mating and put into a centrifugal precipitation tube. 2.0 ml of the deproteinizing agent in D tube was added into the precipitation tube, and the content was stirred thoroughly with a glass rod,

followed by centrifuging. Then the supernatant liquid was separated.

2 1.0 Milli-liter of the supernatant liquid of (lwas,

placed in a test tube and neutralized with 5 6 drops of the neutralizing agent in E tube.

that is, exhibited agglutination of blood cells.

3 0.1 Milli-liter of the neutralized test sample of (2) I was added into A tube to dissolve the antiserum.

4 1.0 Milli-liter of the phosphoric acid buffer solution in C tube was added into B tube to make a i PMS-sensitized blood cell suspension. 5. 0.2 Milli-liter of the sensitized blood cell suspension of (4) was added to A- tube of (3), well shaken, and left to stand on a rack. 6.;After approximately 2 hours standing, the state of aggregate. formed on the bottom of A tube was observed for, the diagnosis. 1

As the result, no agglutination of blood cells was observed, but the erythrocytes sedimented in a ring form was observed. Y I

The same test was given to 113 testsamples taken from pregnant mares on the 45th to80th days after mating, with'correctly the positive reaction results. The accuracy of the diagnosis was 95.9 percent. Also when the test was performed as to 33 non-pregnant mares, the re'sultswere negative with 100 percent accuracy.

EXAMPLE 2 Each 2 ml of serums of pregnant and non-pregnant mares were placed in small test tubes, and added with 2 ml of 0.35 M perchloric acid solution. After thorough shaking, the test tubes were either subjected to a centrifuge at a rate of approximately 3,000 rpm. for 10 minutes, or left standing in a refrigerator for at least three hours. whereupon the coagulated proteins were precipitated. The supernatant liquids or slightly turbid the similar testing procedures as in the above, all the test samples exhibited positive reaction results.

EXAMPLE 5 Using theset-of diagnostic chemicals specified in Example l, assay was similarly performed on the test samples of the maresserums taken on various dates from mating-The results were as given in the table below. Thus the positive reaction appeared in the test samples from the serums extracted from the mares on the 39th to46th days after mating. The positive reaction of the test samples continued until 71 104 days after fecun- I dation of the mares. 1

Fooling Days after last mating v Pedigree Age record and results of immunoassay Percheron 6 i9 2 8 39 49 58,69 79 100 129 Breton 8 4 21 30 41 51 60 71 81 102 129 L 4 f'-i-"+ it Breton l0 4 36 46 55 66 76 97 I26 Anglo-norman s 3 2s 32 43 5:1 62 73 as 104 131 Trotter 4 1 2o 31 41 so 61 71 91121 ExampleS Heavy mere 5 Light mare Results of immunoassay Results otimmunoassay i V Number Accuracy, Number Accuracy, Number of days after mating 0! tests percent of tests percent 1 0 1 0 12 0 12 o 9 6 3 66.7 v 46 25, 21 54.3 '36 1 97.2 61 59 2 96.7 414 0 100.0 11 5 6 45.5 121 u, 5 1 4 20.0

1 Light mare; Thoroughbred, Arab, Anglo-arab. 2 Heavy mare: Percheron, Breton.

What is claimed is:

liquids in the upper layer were taken with a pipette, neutralized, and: subjected to the haemagglutination test. Test samples prepared from pregnant mares serum showed positive reaction, i.e., showed no agglutination of blood cells. Whereas, test samples from non- 5 pregnant mares serum exhibited negative reaction,

EXAMPLE 3 To each 2 ml of serums of pregnant and nonliquids were assayed in the manner similar to the procedure described in Example 1. The test samples from pregnant mares showed positive reaction, and

those from non-pregnant mares, negative reaction.

However, when the above metaphosphoric acid solution was left to stand for a week and thereafter used in 1. In a method of diagnosis of pregnancy of a mare comprising mixing a test sample prepared from blood, plasma or serum containing pregnant mare serum gonadotrophin but substantially no pr'oteinous impurities with antiserum against pregnant mare serum gonadotrophin; mixing the resultant solution with blood cells sensitized with pregnant mare serum 52 orifi filgfiega ut1nat1on inhtbition reaction test,-

sample in a liquid state by (1) adding sulfosalicyclic acid to blood, plasma, or serum of the mare to be diagnosed at a concentration ranging from 0.2 to 1.2 m-mol based on 1 ml of serum and in an amount of 0.5 to 2 volume times the serum;- (2) separating the resultant precipitate; (3) recovering the supernatant liquid, and r (4) neutralizing the recovered supernatant liquid with an alkali to a pH within the range of 3 to 9.5.

lowing the solution to stand to' the improvement which comprises preparing the test 

